The overall goal of the proposed work is to bridge the gap between biochemical rate constants measured in vitro and physiological function. The means by which this gap can be bridged is a computational model that recapitulates IKB/NF-KB signaling and genetic complementation to study the behavior of in vitro characterized mutants. Our goal is to relate biochemical and biophysical findings by co-PI's laboratories to physiologically relevant signal transduction characteristics, and to motivate their work by providing context and focus. The specific aims are: (1) Little is known about the degradation mechanism of free (not NF-KB-bound) IKB protein despite its enormous effect on the signaling characteristics of the pathway. We will use an unbiased proteomic approach to characterize the free IKB polypeptide and potential degradation intermediates. These studies will provide context and inform subsequent work. (2) Mutant IKBa proteins previously characterized in vitro, will be examined in vivo. These studies will correlate dynamic compact foldedness, thermal stability, proteasome sensitivity and in vivo degradation rates, thereby revealing which characteristics are important in IKBa half-life control. (3) Such mutants will be used to study the functional role of interaction and degradation rate constants in signaling. To that end we will construct a lentiviral complementation system that allows for faithful expression in IKB knockout cells. These studies will be guided by computational simulations to investigate the signal transduction characteristics of the IKB/NF-KB signaling module. (4) IKBa is thought to mediate post-induction repression of gene expression by removing NF-KB from promoter DNA. Regulated folding of sequences in the p65 protein appear to play a role in vitro. Using knockout cell lines, complemented with appropriate mutants, we will investigate this process guided by in vitro studies. These studies will culminate with complementation of p65 knockout mice by lentiviral transgenesis.